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Proteoform Identification Using Multiplexed Top-Down Mass Spectra.

Zhige Wang1, Xingzhao Xiong2, Xiaowen Liu2

  • 1Department of Computer Science, Tulane University, New Orleans, Louisiana, USA.

Proteomics
|July 31, 2025
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Summary

Top-down mass spectrometry (TDMS) identifies proteoforms. A new tool, TopMPI, enhances identification of multiple co-fragmented proteoforms in complex tandem mass spectrometry (MS/MS) spectra, improving accuracy and sensitivity.

Keywords:
database searchingmultiplexingproteoform identificationtop‐down proteomics

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Computational Biology

Background:

  • Top-down mass spectrometry (TDMS) is crucial for analyzing intact proteoforms, including posttranslational modifications and sequence variations.
  • Multiplexed TD-MS/MS spectra, arising from co-fragmented proteoforms, present significant challenges for accurate identification and quantification.
  • Existing computational tools struggle with the complexity of multiplexed TD-MS/MS spectra.

Purpose of the Study:

  • To develop a novel computational tool, TopMPI, specifically designed for the identification of multiplexed TD-MS/MS spectra.
  • To enhance the sensitivity and accuracy of proteoform identification in complex MS/MS datasets.
  • To facilitate the discovery of low-abundance proteoforms and potential biomarkers.

Main Methods:

  • Development of TopMPI, a computational tool utilizing a two-round database search strategy.
  • Application of TopMPI to analyze multiplexed TD-MS/MS spectra.
  • Comparative analysis of TopMPI's performance against existing proteoform identification tools.

Main Results:

  • TopMPI significantly increases the sensitivity of proteoform identification in multiplexed TD-MS/MS spectra.
  • TopMPI demonstrates improved accuracy in identifying co-fragmented proteoforms compared to current methods.
  • Experimental results validate TopMPI's effectiveness in handling complex spectral data.

Conclusions:

  • TopMPI represents a substantial advancement in the analysis of multiplexed TD-MS/MS spectra.
  • The tool enhances the capability of TDMS for identifying low-abundance proteoforms in complex biological samples.
  • TopMPI holds promise for advancing proteoform biomarker discovery in disease studies.